oxadiazoles and Respiratory-Distress-Syndrome

Soluble guanylyl cyclase (sGC) is a cGMP-generating enzyme implicated in the control of smooth muscle tone that also regulates platelet aggregation. Moreover, sGC activation has been shown to reduce leukocyte adherence to the endothelium. Herein, we investigated the expression of sGC in a murine model of LPS-induced lung injury and evaluated the effects of sGC inhibition in the context of acute lung injury (ALI). Lung tissue sGC alpha1 and beta1 subunit protein levels were determined by Western blot and immunohistochemistry, and steady-state mRNA levels for the beta1 subunit were assessed by real-time PCR. LPS inhalation resulted in a decrease in beta1 mRNA levels, as well as a reduction in both sGC subunit protein levels. Decreased alpha1 and beta1 expression was observed in bronchial smooth muscle and epithelial cells. TNF-alpha was required for the LPS-triggered reduction in sGC protein levels, as no change in alpha1 and beta1 levels was observed in TNF-alpha knockout mice. To determine the effects of sGC blockade in LPS-induced lung injury, mice were exposed to 1H-[1,2,4]oxodiazolo[4,3-a]quinoxalin-l-one (ODQ) prior to the LPS challenge. Such pretreatment led to a further increase in total cell number (mainly due to an increase in neutrophils) and protein concentration in the bronchoalveoalar lavage fluid; the effects of ODQ were reversed by a cell-permeable cGMP analog. We conclude that sGC expression is reduced in LPS-induced lung injury, while inhibition of the enzyme with ODQ worsens lung inflammation, suggesting that sGC exerts a protective role in ALI.

Topics: Aerosols; Animals; Blotting, Western; Bronchi; Bronchoalveolar Lavage Fluid; Cyclic GMP; Enzyme Inhibitors; Epithelial Cells; Guanylate Cyclase; Immunohistochemistry; Inflammation; Inhalation; Lipopolysaccharides; Lung Injury; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Muscle, Smooth; Oxadiazoles; Protein Subunits; Quinoxalines; Respiratory Distress Syndrome; RNA, Messenger; Solubility; Tumor Necrosis Factor-alpha

Sepsis and posttraumatic inflammatory processes are accompanied by definite changes in microvascular permeability, particularly in the lung. These permeability changes may occur because of damaged regulatory mechanisms at the level of the capillary wall. Pericytes are adventitial cells located within the basement membrane of capillaries. These cells contain multiple cytoplasmic processes that envelope endothelial cells, and are consequently thought to stabilize capillary walls and participate in microcirculation and endothelial cell permeability. Data from this laboratory and other laboratories have confirmed that pericytes are contractile cells, adding to the evidence that pericytes may influence or help regulate capillary permeability. We have already determined that hydrogen peroxide (H2O2) causes dose-dependent relaxation in microvascular lung pericytes (MLPs) at 10 minutes and, conversely, dose-dependent contraction at 30 minutes. It is the aim of this study to determine the mechanism of this biphasic contractile response. Specifically, we will determine whether cyclic adenosine monophosphate (cAMP)- or cyclic guanosine monophosphate (cGMP)-dependent protein kinase intracellular pathways are responsible for the hydrogen peroxide-induced contractility of MLPs.. Rat MLPs were isolated by previously published protocol and cultured on collagen gel matrices. MLPs were pretreated with either ODQ, a soluble guanylate cyclase inhibitor (100 mumol/L), for 15 minutes; GKIP, a protein kinase G inhibitor (100 mumol/L), for 1 hour; SQ22536, an adenylate cyclase inhibitor (100 mumol/L), for 15 minutes; or H89, a protein kinase A inhibitor (10 mumol/L), for 1 hour. Hydrogen peroxide was then introduced to each MLP culture at 10 mumol/L, 100 mumol/L, and 1 mmol/L. After each of these treatments, the surface area of the collagen gels was digitally quantified at 10 and 30 minutes.. SQ22536 attenuated both relaxation at 10 minutes and the contraction seen at 30 minutes for all concentrations of H2O2. H89 caused a marked basal relaxation and prevented the cells from contracting at 30-minute exposures to all concentrations of H2O2. Both ODQ and GKIP attenuated the relaxation at 10 minutes but had no affect on the later contraction.. The cGMP-dependent protein kinase pathway is a mechanism for H2O2-induced relaxation of MLPs. Up-regulation of cAMP and cGMP is responsible for early H2O2-induced relaxation and late contraction. Protein kinase A (cAMP-dependent protein kinase pathway) may be an important intracellular signaling protein in the H2O2-induced contraction of MLPs or may be unable to down-regulate cAMP once inhibited. This evidence further supports the concept that there are separate intracellular pathways that regulate divergent cellular responses. This idea parallels the clinical concept of reversible and irreversible dysfunction of cellular processes in shock, and that the cellular dysfunction is initiated by separate intracellular pathways.

Topics: Adenine; Adenylyl Cyclase Inhibitors; Analysis of Variance; Animals; Cell Survival; Cells, Cultured; Cyclic AMP-Dependent Protein Kinases; Enzyme Inhibitors; Hydrogen Peroxide; Isoquinolines; Lung; Male; Muscle Contraction; Oxadiazoles; Pericytes; Quinoxalines; Rats; Rats, Sprague-Dawley; Respiratory Distress Syndrome; Sepsis; Sulfonamides